Antibody conjugates of cytotoxic drugs are being developed as target-specific therapeutic agents. Antibodies against various cancer cell-surface antigens have been conjugated with different cytotoxic agents (see Chari 1998, Adv. Drug Delivery Revs., 31, p 89-104; Chari, 2008, Acc. Chem. Res., 41, p 98-107; Ducry & Stump, 2010, Bioconjugate Chem., 21, p 5-13; Senter, 2009, Curr Opinions in Chem. Biol., 13, p 235-244; Ojima. et al., 2002, J. Med. Chem. 45, 5620-5623; Hamann, P. R. et al., 2002, Bioconjug Chem. 13, 47-58; Bross, P. F. et al., 2001 Clin Cancer Res. 7, 1490-6; DiJoseph, J. F. et al., 2004, Blood, 103:1807-1814; Doronina, S. O., et al., 2003, Nat. Biotechnol. 21, 778-784; Doronina, S. O., et al., 2006, Bioconjug Chem. 17, 114-24).
The cytotoxic compounds used in antibody-drug conjugates inhibit various essential cellular targets, such as microtubules (maytansinoids, auristatins, taxanes: U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333,410; 6,441,163; 6,340,701; 6,372,738; 6,436,931; 6,596,757; 7,276,497; 7,301,019; 7,303,749; 7,368,565; 7,473,796; 7,585,857; 7,598,290; 7,495,114; 7,601,354, U.S. Patent Application Nos. 20100092495, 20100129314, 20090274713, 20090076263, 20080171865) and DNA (calicheamicin, doxorubicin, CC-1065 analogues: U.S. Pat. Nos. 5,475,092; 5,585,499; 5,846,545; 6,534,660; 6,756,397; 6,630,579; 7,388,026; 7,655,660; 7,655,661). In these conjugates, the cytotoxic moiety and the antibody are linked together either via a cleavable linker, such as a disulfide bond, acid-labile bond, peptidase-labile bond, or esterase-labile bond, or via a non-cleavable linker, such as a thioether bond or an amide bond. Antibody conjugates with some of these cytotoxic drugs are actively being investigated in the clinic for cancer therapy (Richart, A. D., and Tolcher, A. W., 2007, Nature Clinical Practice, 4, 245-255; Krop et al., 2010, J. Clin. Oncol., 28, p 2698-2704).
A wide array of linker technologies has been employed for the preparation of such immunoconjugates, and both cleavable and non-cleavable linkers have been investigated. In most cases, the full cytotoxic potential of the drugs could only be observed, however, if the drug molecules could be released from the conjugates in unmodified form at the target site.
One of the cleavable linkers that has been employed for the preparation of antibody-drug conjugates is an acid-labile linker based on cis-aconitic acid that takes advantage of the acidic environment of different intracellular compartments such as the endosomes encountered during receptor mediated endocytosis and the lysosomes. Shen and Ryser introduced this method for the preparation of conjugates of daunorubicin with macromolecular carriers (Biochem. Biophys. Res. Commun. 102:1048-1054 (1981)). Yang and Reisfeld used the same technique to conjugate daunorubicin to an anti-melanoma antibody (J. Natl. Canc. Inst. 80:1154-1159 (1988)). Dillman et al. used an acid-labile linker in a similar fashion to prepare conjugates of daunorubicin with an anti-T cell antibody (Cancer Res. 48:6097-6102 (1988)).
An alternative approach, explored by Trouet et al. involved linking daunorubicin to an antibody via a peptide spacer arm (Proc. Natl. Acad. Sci. 79:626-629 (1982)). This was done under the premise that free drug could be released from such a conjugate by the action of lysosomal peptidases.
Maytansinoids are highly cytotoxic drugs. Maytansine was first isolated by Kupchan et al. from the east African shrub Maytenus serrata and shown to be 100 to 1000-fold more cytotoxic than conventional cancer chemotherapeutic agents like methotrexate, daunorubicin, and vincristine (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that some microbes also produce maytansinoids, such as maytansinol and C-3 esters of maytansinol (U.S. Pat. No. 4,151,042). Synthetic C-3 esters of maytansinol and analogues of maytansinol have also been reported (Kupchan et al. J. Med. Chem. 21:31-37 (1978); Higashide et al. Nature 270:721-722 (1977); Kawai et al. Chem. Pharm. Bull. 32:3441-3451 (1984)). Examples of analogues of maytansinol from which C-3 esters have been prepared include maytansinol with modifications on the aromatic ring (e.g. dechloro) or at the C-9, C-14 (e.g. hydroxylated methyl group), C-15, C-18, C-20 and C-4,5.
The naturally occurring and synthetic C-3 esters of maytansinol can be classified into two groups:                (a) C-3 esters with simple carboxylic acids (U.S. Pat. Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598 and Chem. Pharm. Bull. 1984, 12:3441), and        (b) C-3 esters with derivatives of N-methyl-L-alanine (U.S. Pat. Nos. 4,137,230; 4,260,608; 5,208,020; and Chem. Pharm. Bull. 1984, 12:3441).        
Esters of group (b) bearing an acylated N-methylalanine ester were found to be much more cytotoxic than the ansamitocin esters of group (a). For example, Kupchan et al. (J. Med. Chem., 21; 31 (1978) reported that ansamitocin analogues (simple C3 esters of maytansinol) such as a propionyl ester, bromoacetyl ester, crotonyl ester and trifluoroacetyl ester were 34 to 1640-fold less potent (IC50=0.00021−0.01 micrograms/mL) towards cancer cells than esters such as maytansine that bear an acylated N-methyl-L-alanine ester at C3 (IC50=0.0000061 micrograms/mL). Of the six simple ansamitocin esters tested in this study only 2 of them had comparable potency to the N-methylalanine containing compound maytansine. In contrast, N-acyl-N-methyl-L-alanyl esters of maytansinol were highly potent regardless of the nature of the acyl group. Other modified ansamitocins have been reported by Kawai et al. (Chem. Pharm. Bull., 32; 3441, 1984). Most of these compounds were reported to have anti-microbial activity at 1 to 4 micrograms/mL. Cell killing activity towards cancer cells in vitro was not reported.
Maytansine is an N-acetyl-N-methyl-L-alanyl ester of maytansinol. It is a highly potent mitotic inhibitor. Treatment of L1210 cells in vivo with maytansine has been reported to result in 67% of the cells accumulating in mitosis. Untreated control cells were reported to demonstrate a mitotic index ranging from between 3.2 to 5.8% (Sieber et al. 43 Comparative Leukemia Research 1975, Bibl. Haemat. 495-500 (1976)). Experiments with sea urchin eggs and clam eggs have suggested that maytansine inhibits mitosis by interfering with the formation of microtubules through the inhibition of the polymerization of the microtubule protein, tubulin (Remillard et al. Science 189:1002-1005 (1975)).
In vitro, P388, L1210, and LY5178 murine leukemic cell suspensions have been found to be inhibited by maytansine at doses of 10−3 to 10−1 μg/μl, with the P388 line being the most sensitive. Maytansine has also been shown to be an active inhibitor of in vitro growth of human nasopharyngeal carcinoma cells, and the human acute lymphoblastic leukemia line CEM was reported inhibited by concentrations as low as 10−7 mg/ml (Wolpert-DeFillippes et al. Biochem. Pharmacol. 24:1735-1738 (1975)).
In vivo, maytansine has also been shown to be active. Tumor growth in the P388 lymphocytic leukemia system was shown to be inhibited over a 50 to 100-fold dosage range, which suggested a high therapeutic index; also significant inhibitory activity could be demonstrated with the L1210 mouse leukemia system, the human Lewis lung carcinoma system and the human B-16 melanocarcinoma system (Kupchan, Fed. Proc. 33:2288-2295 (1974)).
Based on its high potency, analogues of maytansine bearing various acyl side chains on the N-methylalanyl moiety suitable for linking to cell binding agents have been described (see for example U.S. Pat. Nos. 5,208,020; 5,416,064; 7,473,796; 7,368,565; 7,301,019; 7,276,497; 6,716,821; 6,441,163; U.S. Patent Application Nos. 20100129314; 20100092495; 20090274713; 20090076263; 20080171865; 20080171856; 20070270585; 20070269447; 20070264266; and 20060167245; Chari et al., Cancer Res., 52: 127-131 (1992); Liu et al., Proc. Natl. Acad. Sci., 93: 8618-8623 (1996); and Widdison et al., J. Med. Chem., 49; 4392, 2006). In these conjugates, the cell-binding agent is linked via disulfide bonds to the maytansinoids such as DM1 [N2′-deacetyl-N-2′(3-mercapto-1-oxopropyl)-maytansine, CAS Number: 139504-50-0, FIG. 1] or DM4 [N2′-deacetyl-N-2′(4-mercapto-4-methyl-1-oxopentyl)-maytansine, CAS Number: 796073-69-3].
In the above patents, the maytansinoid drugs used for linkage to cell binding agents bear an acylated N-methyl alanine or a N-methyl cysteine-containing ester side chain (see FIG. 1). The N-methyl alanine or N-methyl cysteine containing side chain has to be of the L configuration to get high potency, with the D-isomer being up to 100-fold less potent (see Widdison et al., J. Med. Chem., 49; 4392, 2006).